Compositions prepared to contain active dextran sucrase produced by Leuconostoc mesenteroides ssp. cremoris

ABSTRACT

A food or cosmetic composition containing active dextran sucrase enzymes which are produced by a strain of  Leuconostoc mesenteroides  ssp.  cremoris  and which synthesize dextran. In addition to culturing the strain in a food or cosmetic composition containing sucrose to obtain a composition containing the active enzymes, the strain may be cultured in a culture medium containing sucrose to obtain a culture product containing the active dextran sucrase enzymes which may be isolated from the culture product and incorporated into a food or cosmetic substance.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of application Ser. No.09/087,643 which was filed May 29, 1998 and which issued as U.S. Pat.No. 6,004,800.

BACKGROUND OF THE INVENTION

The present invention relates to dextran sucrase produced by bacteriaand means for production of the same and its use.

STATE OF THE ART

Dextran is a polysaccharide formed of glucose units, the chainlengthening of which is catalyzed by dextran sucrase. The biosynthesisof dextran has been demonstrated in numerous bacteria, especially inStreptococcus mutans, Leuconostoc mesenteroides ssp. mesenteroides andLeuconostoc mesenteroides ssp. dextranicum. Leuconostoc produce theenzyme dextran sucrase and secrete it into the culture medium in thepresence of sucrose. This enzyme, dextran sucrase, then synthesizesdextran from the sucrose substrate. Dextran has applications in severalfields. It is used especially in biochemistry as a support forfiltration chromatography on a gel of the Sephadex type. Additionally,in the field of therapeutics, it is used as a substitute for bloodplasma (Biochimie générale (General Biochemistry)—J. H. WEIL—Masson, 6thedition—1990—p. 171).

Furthermore, dextran synthesized by a strain of Leuconostoc dextranicumis applied in the food industry for the texturing of food products suchas yoghurts, cream desserts, milk-based drinks and salad dressings.European Patent Application Publication No. EP 0 363 633 demonstratesthe synthesis of dextran by a strain of Leuconostoc dextranicum and inparticular by the strain Leuconostoc dextranicum NRRL-B-18242.Additionally , that patent application publication describes especiallya composition containing dextran synthesized by this bacterium and theuse of this composition in the food sector.

Moreover, the taxonomy of the bacterial strains of the genus Leuconostochas been revised several times.

Garvie et al. (International Journal of Systematic Bacteriology,118-119, 1983) describe the taxonomy of the bacterial strains of thegenus Leuconostoc, established according to a criterion of homology interms of the deoxyribonucleic acid (DNA). The bacteria, previouslyclassed as Leuconostoc mesenteroides, Leuconostoc dextranicum andLeuconostoc cremoris, although of different phenotype, have a very highdegree of homology in respect of their DNA. Therefore, according to thistaxonomy, these bacteria are subspecies of Leuconostoc mesenteroides andare respectively called Leuconostoc mesenteroides ssp. mesenteroides,Leuconostoc mesenteroides ssp. dextranicum and Leuconostoc mesenteroidesssp. cremoris.

In addition, J. B. Milliere et al. (Journal of Applied Bacteriology, 67,529-542, 1989) describe a taxonomic analysis performed on 81 strains ofthe genus Leuconostoc, including 11 strains of Leuconostoc mesenteroidesssp. cremoris. This analysis relies on the taxonomy established byGarvie et al. and is based on the following criteria in particular: thecapacity of these strains to ferment various sugars, the capacity ofthese strains to utilize citrate and the capacity of these strains toproduce dextran. This document notes the fact that a strain ofLeuconostoc mesenteroides ssp. cremoris does not synthesize dextran.Also, such a strain is distinguished and defined by the fact that itdoes not ferment pentoses.

No strain of Leuconostoc mesenteroides ssp. cremoris has yet beenisolated which is capable of synthesizing dextran. Now, Leuconostocmesenteroides ssp. cremoris is of major importance in the manufacture ofdairy products such as, for example, yoghurt-type fermented specialitiesor dairy creams. It would therefore be very valuable to be able to usesuch bacteria, capable of synthesizing dextran of pleasant texture andtaste, especially for texturing this type of food product.

The object of the present invention is to meet this need.

SUMMARY OF THE INVENTION

For meeting the need noted above the present invention provides strainsof Leuconostoc mesenteroides ssp. cremoris which produce dextran,especially the strains Leuconostoc mesenteroides ssp. cremoris CNCMI-1692 and CNCM I-1693.

The present invention further provides a method of using a strain ofLeuconostoc mesenteroides ssp. cremoris for the manufacture of a foodproduct or cosmetic composition.

The present invention further provides a process for the production ofdextran from Leuconostoc mesenteroides ssp. cremoris.

The present invention further provides a food composition or cosmeticcomposition into which dextran obtained by carrying out the process isincorporated during its preparation.

The present invention further provides a process for the production ofan additive containing active dextran sucrase from Leuconostocmesenteroides ssp. cremoris.

The present invention finally relates to a food product or cosmeticcomposition which contains active dextran sucrase produced byLeuconostoc mesenteroides ssp. including a composition into which anadditive containing active dextran sucrase from Leuconostocmesenteroides ssp. cremoris is incorporated during its preparation.

DETAILED DESCRIPTION OF THE INVENTION

As described above, present invention provides strains of Leuconostocmesenteroides ssp. cremoris which produce dextran. It was possible toisolate strains of Leuconostoc mesenteroides ssp. cremoris which producedextran. All the strains of Leuconostoc mesenteroides ssp. cremoriswhich produce dextran are therefore covered by the present invention.

A strain of Leuconostoc mesenteroides ssp. cremoris was isolatedparticularly from a Swiss cream and it was found, surprisingly, to havethe remarkable property of synthesizing dextran of pleasant texture andtaste. This strain was deposited on Apr. 18, 1996, under the terms ofthe Budapest Treaty, in the Collection National de Cultures deMicroorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724PARIS CEDEX 15, where it was given the deposit number CNCM I-1692.

Furthermore, a strain of Leuconostoc mesenteroides ssp. cremoris whichalso has the remarkable property of synthesizing dextran of pleasanttexture and taste was isolated by natural selection from the strain CNCMI-1692. This strain was deposited on Apr. 4, 1996, under the terms ofthe Budapest Treaty, in the Collection Nationale de Cultures deMicroorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724PARIS CEDEX 15, where it was given the deposit number CNCM I-1693.

Details of these strains, concerning especially their morphology, thefermentation of sugars and other aspects, are given below.

Morphology

Gram-positive microorganisms,

Negative catalase,

Facultative aerobe, and

Cocci.

Fermentation of Sugars

No lactic acid production from pentoses, D- and L-arabinose, D- andL-xylose and D- and L-ribose,

Lactic acid production from lactose by the strain CNCM I-1692, and

No lactic acid production from lactose by the strain CNCM I-1693.

Other Aspects

Strains synthesizing dextran, a polysaccharide with remarkable texturingproperties.

According to the present invention, it is therefore possible also toisolate a strain of Leuconostoc mesenteroides ssp. cremoris which doesnot ferment lactose, an example being CNCM I-1693.

A preferred strain according to the present invention produces the samedextran as the strain CNCM I-1692 or the strain CNCM I-1693.

As indicated above, present invention provides a process for theproduction of dextran wherein a medium containing sucrose is inoculatedwith a preculture of a strain of Leuconostoc mesenteroides ssp. cremorisaccording to the invention, it is allowed to ferment at 25-35° C. for10-20 h and the pH of the resulting culture is then lowered to 5-5.5prior to storage at 0-10° C. for 16-48 h.

A medium containing at least 2% of sucrose can be inoculated with apreculture of a strain of Leuconostoc mesenteroides ssp. cremorisaccording to the invention, for example in order to allow the productionof dextran sucrase and the synthesis of dextran in the culture medium.

A medium containing 5-12% of MSK medium (skimmed cow's milk)supplemented with 0.05-0.2% of yeast extract and at least 2% of sucrose,for example, can be inoculated with 0.2-3% of a preculture of a strainof Leuconostoc mesenteroides ssp. cremoris according to the presentinvention, particularly the strain CNCM I-1692 or the strain CNCMI-1693. The medium can be allowed to ferment at 25-35° C. for 10-20 hwith the pH being maintained at 6-7.3, for example. Then, whenfermentation has ended, the pH of the resulting culture can be loweredto 5-5.5 by the addition of lactic acid, for example. The culture isthen stored at 0-10° C. for 16-48 h.

Also, a medium containing 0.05-0.2% of yeast extract with at least 2% ofsucrose, for example, can be inoculated with 0.2-3% of a preculture of astrain of Leuconostoc mesenteroides ssp. cremoris according to thepresent invention, particularly the strain CNCM I-1692 or the strainCNCM I-1693. The medium can be allowed to ferment at 25-35° C. for 10-20h with the pH being maintained at 6-7.3, for example. When fermentationhas ended, this culture can be mixed with an equal volume of 20% MSKmedium so that inhibition of the production of the enzyme dextransucrase by the lactose contained in the MSK medium, for example, isavoided during the fermentation. The pH of the resulting culture canthen be lowered to 5-5.5 by the addition of lactic acid, for example.The culture is then stored at 0-10° C. for 16-48 h.

This culture can then be dried to give a dextran powder, for example.This culture can be dried by lyophilization or spray drying, forexample.

As indicated above, present invention further provides a food product orcosmetic composition comprising dextran from Leuconostoc mesenteroidesssp. cremoris which is obtainable by carrying out the above process.

To prepare such a product or composition, dextran obtained by carryingout the above process for the production of dextran can be incorporatedinto a food or cosmetic product, such as a milk powder, a yoghurt, aketchup, a mayonnaise or a skin cream, during its manufacture, forexample.

As also indicated above, present invention further provides a processfor the production of an additive containing active dextran sucrasewherein a medium containing sucrose is inoculated with a preculture of astrain of Leuconostoc mesenteroides ssp. cremoris according to theinvention and is then allowed to ferment at 25-35° C. for 10-20 h.

A medium containing at least 2% of sucrose can be inoculated with apreculture of a strain of Leuconostoc mesenteroides ssp. cremorisaccording to the invention in order to allow the production of dextransucrase in the culture medium, for example.

A medium containing 5-12% of MSK medium (skimmed cow's milk)supplemented with 0.05-0.2% of yeast extract and at least 2% of sucrose,for example, can be inoculated with 0.2-3% of a preculture of a strainof Leuconostoc mesenteroides ssp. cremoris according to the presentinvention, particularly with 0.2-3% of a preculture of the strain CNCMI-1692 or the strain CNCM I-1693. It can be allowed to ferment at 25-35°C. for 10-20 h with the pH being maintained at 6-7.3, for example.

A medium containing 0.05-0.2% of yeast extract and at least 2% ofsucrose, for example, can be inoculated with 0.2-3% of a preculture of astrain of Leuconostoc mesenteroides ssp. cremoris according to thepresent invention, particularly with 0.2-3% of a preculture of thestrain CNCM I-1692 or the strain CNCM I-1693. It can be allowed toferment at 25-35° C. for 10-20 h with the pH being maintained at 6-7.3,for example. When fermentation has ended, this culture can be mixed withan equal volume of 20% MSK medium so that inhibition of the productionof the enzyme dextran sucrase by the lactose contained in the MSKmedium, for example, can be avoided during the fermentation.

Also, a synthetic culture medium containing at least 2% of sucrose, 1-3%of K₂HPO₄, 0.2-1% of yeast extract, 0.2-1% of peptone and 0.0005-0.001%of MnSO₄ can be inoculated with 0.2-3% of a preculture of a strain ofLeuconostoc mesenteroides ssp. cremoris according to the presentinvention, particularly with 0.2-3% of a preculture of the strain CNCMI-1692 or the strain CNCM I-1693. It can be allowed to ferment at 25-35°C. for 7-12 h with the pH being maintained at 6-7.3, for example.

In a first preferred embodiment of the process for the preparation of anadditive containing active dextran sucrase, the pH of the culture isadjusted to 5-5.5 after fermentation and this culture is then dried togive a powder containing the active dextran sucrase. The pH of theculture can be adjusted by the addition of lactic acid, for example. Theculture can be dried by lyophilization or by spray drying, for example.

In a second preferred embodiment of the process for the preparation ofactive dextran sucrase, the culture is separated after fermentation soas to isolate the supernatant containing the active dextran sucrase.This separation can be effected by centrifugation at 15,000-20,000 g for10-35 min at 2-6° C., for example.

It is possible to adjust the pH of the supernatant to 4.9-5.7, storethis supernatant at 0-10° C. for 15-30 h and then precipitate themacromolecules contained in this supernatant so as to isolate aprecipitate containing the dextran sucrase, for example. Themacromolecules in the supernatant can be precipitated with polyethyleneglycol or ammonium sulphate at 2-6° C., with stirring, for example.

The precipitate containing the dextran sucrase can then be dialyzed soas to eliminate the precipitating agents, for example.

Finally, in this second preferred embodiment of the present process, theprecipitate containing the dextran sucrase can be stored at atemperature below −4° C. after the precipitation step or after thedialysis step, for example.

As indicated above, present invention further provides a food product orcosmetic composition comprising an additive containing active dextransucrase from Leuconostoc mesenteroides ssp. cremoris obtainable bycarrying out the above process.

To prepare such a product or composition, an additive containing activedextran sucrase obtained in this way can be incorporated into a food orcosmetic product, such as a milk powder, a yoghurt, a ketchup, amayonnaise or a skin cream, during its manufacture, for example.

Finally, the present invention further provides to a method of using astrain of Leuconostoc mesenteroides ssp. cremoris according to thepresent invention for the manufacture of a food product or cosmeticcomposition.

The strains of Leuconostoc mesenteroides ssp. cremoris, the dextransucrase produced by these strains and the dextran synthesized by thesestrains according to the present invention are characterized in greaterdetail below by means of different microbiological and biochemical dataillustrating their properties. The percentages are given by weight,unless indicated otherwise.

METHODOLOGIES

Testing for the Strains of Leuconostoc which Produce Dextran

Tests for the strains which produce dextran were performed on 150strains isolated either from dairy products or from non-dairy productssuch as wine, coffee and sauerkraut.

The production of dextran in a medium containing sucrose is measured.

To do this, 10 ml of DEX medium comprising 1% of B. tryptone, 0.5% ofyeast extract, 0.5% of K₂HPO₄, 0.5% of ammonium citrate and 5% ofsucrose are inoculated with 1% of a preculture of each of the 150strains. The medium is then allowed to ferment at 30° C. for 24 h.

16 strains capable of producing dextran were thus selected from the 150starting strains.

150 ml of DEX medium, as described above, are then inoculated with 1% ofa preculture of each of these 16 selected strains and then allowed toferment at 30° C. for 24 h prior to measurement of the viscosity of theproduct of these 16 cultures obtained in this way. The viscosity ismeasured with a gravity viscometer of diameter 25 mm.

Table I below shows the values in seconds for the passage of 100 ml ofthe product of each culture through the gravity viscometer. The strainA1, shown in Table I, was used as a negative control strain.

In Table I, the strains A are strains of Leuconostoc sp., the strains Bare strains of Leuconostoc mesenteroides ssp. cremoris, the strain C1 isa strain of Leuconostoc lactis and the strains D are strains ofLeuconostoc mesenteroides spp. mesenteroides.

TABLE I Strain Viscosity (s) A1 11 A2 16 A3 14 A4 13 A5 18 A6 20 A7 19A8 19 C1 17 CNCM I-1692 22 B1 12 B2 14 B3 15 D1 12 D2 14 D3 15 D4 17

The results shown in Table I demonstrate the fact that the strainLeuconostoc mesenteroides ssp. cremoris CNCM I-1692 has the highestviscosity of the 16 selected strains, so it is acknowledged that thestrain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692 produces thegreatest amount of dextran.

Study of the Concentration of the Enzyme Dextran Sucrase as a Functionof Fermentation Time

A synthetic medium containing 2% of sucrose, 2% of K₂HPO₄, 0.5% of yeastextract, 0.5% of peptone, 0.02% of MgSO₄, 0.001% of MnSO₄, 0.001% ofFeSO₄ and 0.001% of NaCl is inoculated with 1% of a preculture of thestrain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692. It isallowed to ferment at 30° C. for 12 h in a 5 1 fermenter.

After 4 h of fermentation, a sample of the resulting culture is takenevery 2 hours, up to a fermentation time of 12 h, for measurement of thegrowth of the strain by means of the optical density at 600 nm.

Each sample is then centrifuged at 18,000 g for 20 min at 4° C., thesupernatant is recovered, its pH is adjusted to 5.2 and the activity ofthe dextran sucrase contained in the supernatant is verified bymeasurement of the incorporation of radioactivity into the dextran fromradioactive sucrose (J. Dent. Res. 1974, 53, 1355-1360).

Table II shows the results obtained for measurement of the growth of thestrain on the basis of samples taken every 2 hours between fermentationtimes of 4 and 12 h. Table II also shows the results of measurement ofthe activity of the dextran sucrase contained in the supernatant ofthese samples.

TABLE II fermentation time (h) 4 6 8 10 12 growth (OD₆₀₀)/10 0.034 0.0850.21 0.51 0.53 activity (u/ml) — 0.1 0.17 0.6 0.61

The results given in Table II demonstrate the fact that theconcentration of dextran sucrase reaches its maximum value just afterthe Leuconostoc mesenteroides ssp. cremoris bacteria enter thestationary phase.

Purification of the Enzyme Dextran Sucrase and Determination of itsSpecific Activity

A culture medium containing 0.2% of sucrose, 0.5% of yeast extract, 0.5%of peptone, 2% of K₂HPO₄, 0.02% of MgSO₄, 0.001% of MnSO₄, 0.001% ofFeSO₄ and 0.001% of NaCl is inoculated with 1% of a preculture ofLeuconostoc mesenteroides ssp. cremoris CNCM I-1692 and allowed toferment at room temperature for 12 h with the pH being maintained at avalue of 6.7.

The culture prepared in this way is then centrifuged at 18,000 g for 20min at 4° C.

The supernatant containing the dextran sucrase is then isolated and itspH is adjusted to a value of 5.2 prior to incubation for 4 h at 4° C.

The supernatant is then mixed with an equal volume of 33% polyethyleneglycol 400 and the mixture is allowed to incubate at 4° C. for 5 h, withstirring, so as to precipitate the proteins contained in thesupernatant.

The mixture is centrifuged at 18,000 g for 20 min at 40° C. so as toisolate the residue containing the precipitated proteins.

This residue containing the precipitated proteins is then suspended in70 ml of 20 mM ammonium acetate, pH 5.2.

600 μg of dextranase are then added to this suspension and the whole isallowed to incubate at 25° C. for 1 h so that the dextran contained inthe suspension is digested by the dextranase.

The suspension is dialyzed in the presence of 70 ml of 20 mM ammoniumacetate, pH 5.2, so as to eliminate the glucose molecules obtained afterdigestion of the dextran with the dextranase. The proteins are thenisolated on an anion exchange column (Fast Q, Pharmacia Biotech AB,Uppsala, SU) which has been equilibrated beforehand with a 20 mMammonium acetate buffer, pH 5.2.

The proteins are eluted over a linear gradient of 0-0.5 M NaCl.

The activity of the dextran sucrase in the different protein fractionseluted in this way is analyzed and these protein fractions are subjectedto electrophoresis on an SDS-polyacrylamide gel.

The protein fractions containing the dextran sucrase are isolated andthe activity of the purified dextran sucrase is measured and found to be105 u/mg.

Texturing Capacity of the Dextrans as a Function of the IncubationTemperature of the Enzyme

The activity of the dextran sucrase from the strain Leuconostocmesenteroides ssp. cremoris CNCM I-1692 is demonstrated, on the one handat 30° C. and on the other hand at 4° C., thereby verifying thetexturing capacity of the dextrans at these different temperatures.

This is done by preparing an additive containing active dextran sucrasefrom a culture of the strain Leuconostoc mesenteroides ssp. cremorisCNCM I-1692.

A synthetic medium containing 2% of sucrose, 2% of K₂HPO₄, 0.5% of yeastextract, 0.5% of peptone, 0.02% of MgSO₄, 0.001% of MnSO₄, 0.001% ofFeSO₄ and 0.001% of NaCl is inoculated with 1% of a preculture of thestrain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692. It isallowed to ferment at 30° C. for 12 h with the pH being maintained at6.7.

The culture is then separated by centrifugation at 18,000 g for 20 minat 4° C. so as to isolate the supernatant containing the dextransucrase.

The pH of the supernatant is lowered to 5.2 and this supernatant isincubated for 12 h at 4° C.

The macromolecules in the supernatant are then precipitated twice withpolyethylene glycol at 4° C. so as to purify the dextran sucrase.

The texturing capacity of the dextrans synthesized by the dextransucrase isolated in this way is then verified by incubating the latter,on the one hand at 30° C. and on the other hand at 4° C., in abuffer-substrate containing 20 mM acetate, pH 5.2, 200 mM sucrose and 20mM CaCl₂.

Table III shows the results of texturing with the active dextran sucrasefrom the strain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692, onthe one hand at 30° C. and on the other hand at 4° C.

TABLE III medium incubation conditions texturing a 30° C. for 12 h −  4°C. for 12 h +++++ a: buffer-substrate medium of pH 5.2 −: absence oftexture +++++: good texture, very thick

The results shown in Table III demonstrate the fact that the dextransucrase from the strain Leuconostoc mesenteroides ssp. cremoris CNCMI-1692 makes it possible at 4° C. to synthesize a dextran with a thickand pleasant texture, whereas at 30° C. a turbid solution devoid oftexture is obtained. This difference in texturing is without doubt dueto the fact that at 4° C. the dextran is in the form of molecules withshort branches and thus makes it possible to obtain a thick and pleasanttexture, whereas at 30° C. the dextran is in the form of molecules withlong branches aligned parallel with the main chain. These molecules donot afford a good texture.

Texturing of a Dairy Product with the Dextran Sucrase

The dextran sucrase is incubated under conditions (medium, temperatureand pH) identical to those which prevail during the preparation of ayoghurt.

This is done by preparing an additive containing active dextran sucrasefrom a culture of the strain Leuconostoc mesenteroides ssp. cremorisCNCM I-1692.

A synthetic medium containing 2% of sucrose, 2% of K₂HPO₄, 0.5% of yeastextract, 0.5% of peptone, 0.02% of MgSO₄, 0.001% of MnSO₄, 0.001% ofFeSO₄ and 0.001% of NaCl is inoculated with 1% of a preculture of thestrain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692. It isallowed to ferment at 30° C. overnight with the pH being maintained at6.7.

The culture is then separated by centrifugation at 18,000 g for 20 minat 4° C. so as to isolate the supernatant containing the dextransucrase.

The pH of the supernatant is then lowered to 5.2 and this supernatant isincubated for 12 h at 4° C.

The macromolecules in the supernatant are then precipitated twice withpolyethylene glycol at 4° C. so as to purify the dextran sucrase.

The synthesis of dextran by the dextran sucrase is then verified. Thisis done by incubating the dextran sucrase on the one hand in abuffer-substrate of pH 6.4 containing 20 mM acetate, 200 mM sucrose and20 mM CaCl₂, and on the other hand in a milk drink containing 6% ofsucrose.

Table IV below shows the results obtained for texturing with the dextransucrase from the strain Leuconostoc mesenteroides ssp. cremoris CNCMI-1692.

TABLE IV medium simulation conditions texture a c − d +++++ b c + d+++++ a: buffer-substrate of pH 6.4 b: milk drink containing 6% ofsucrose c: 5 h at 37° C. followed by adjustment of the pH to 4.7,lowering of the temperature to 4° C. and incubation for 24 h at 4° C. d:5 h at 20° C. followed by adjustment of the pH to 4.7, lowering of thetemperature to 4° C. and incubation for 24 h at 4° C. −: absence oftexture +: small amount of texture +++++: good texture

The results shown in Table IV make it possible to demonstrate the factthat the dextran sucrase also textures a milk-based medium, but nolonger produces thickening dextran after 5 h at 37° C. Yoghurts can betextured by adding the dextran sucrase after fermentation and beforestorage at 4° C.

EXAMPLES

The Examples below are given in order to illustrate the use of thedextran, the dextran sucrase and/or a strain producing this dextran andthis dextran sucrase in the manufacture of a food product or cosmeticcomposition according to the present invention. The percentages givenare by weight, unless indicated otherwise.

Example 1

The strain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692 accordingto the present invention is used for the manufacture of yoghurts.

To do this, 1 l of a milk product containing 2.8% of fats andsupplemented with 2% of skimmed milk powder and 6% of sucrose isprepared, it is pasteurized at 96° C. for 30 min and its temperature isthen lowered to 42° C.

In a parallel operation, a frozen preculture of a non-thickening strainof Streptococcus thermophilus and a frozen preculture of a non-viscousstrain Lactobacillus bulgaricus are reactivated in a sterile MSK culturemedium containing 10% of reconstituted milk powder and 0.1% ofcommercial yeast extract.

A frozen preculture of the strain of Leuconostoc mesenteroides ssp.cremoris is also reactivated in an MRS culture medium (MRSlactobacilli—Detroit—USA) and then in a sterile MSK culture mediumcontaining 10% of reconstituted milk powder and 0.1% of commercial yeastextract and supplemented with 1% of sucrose.

The pasteurized milk product is then inoculated with 1% of each of thesereactivated precultures and this milk product is then allowed to fermentat 37° C. until the pH reaches a value of 4.5.

Yoghurts are produced in this way and stored at 4° C.

These yoghurts, prepared with a strain of Leuconostoc mesenteroides ssp.cremoris, have an unctuous texture with a pleasant taste, especiallyafter storage for 10 days at 4° C.

Example 2

The additive containing active dextran sucrase according to the presentinvention is used for the manufacture of yoghurts.

To do this, a synthetic medium containing 2% of sucrose, 2% of K₂HPO₄,0.5% of yeast extract, 0.5% of peptone, 0.02% of MgSO₄, 0.001% of MnSO₄,0.001% of FeSO₄ and 0.001% of NaCl is inoculated with 1% of a precultureof the strain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692. It isallowed to ferment at 30° C. for 12 h with the pH being maintained at6.7. The culture is then separated by centrifugation at 18,000 g for 20min at 4° C. so as to isolate the supernatant containing the activedextran sucrase. The pH of the supernatant is lowered to 5.2 and themacromolecules in the supernatant are then precipitated at 4° C. withammonium sulphate so as to isolate a precipitate containing the dextransucrase. This precipitate is then dialyzed to remove the ammoniumsulphate.

1 l of a milk product containing 2.8% of fats and supplemented with 2%of skimmed milk powder and 6% of sucrose is also prepared, it ispasteurized at 96° C. for 30 min and its temperature is then lowered to42° C.

In a parallel operation, a frozen preculture of a non-thickening strainof Streptococcus thermophilus and a frozen preculture of a non-viscousstrain of Lactobacillus bulgaricus are reactivated in a sterile MSKculture medium containing 10% of reconstituted milk powder and 0.1% ofcommercial yeast extract.

The-pasteurized milk product is inoculated with 1% of each of thecultures of the two strains and the milk product is then incubated at37° C. until the pH reaches a value of 4.5.

1% of purified dextran sucrase is then added, with stirring.

Yoghurts are produced in this way and stored at 4° C.

These yoghurts, prepared with the purified dextran sucrase synthesizedby Leuconostoc mesenteroides ssp. cremoris, have an unctuous texturewith a pleasant taste, especially after storage for 10 days at 4° C.

Example 3

The additive containing active dextran sucrase according to the presentinvention is used in powder form for the manufacture of yoghurts.

To do this, a culture medium containing 9% of skimmed milk powder, 0.1%of yeast extract and 2% of sucrose is inoculated with 1% of a precultureof the strain of Leuconostoc mesenteroides ssp. cremoris. It is allowedto ferment at 30° C. for 20 h with the pH being maintained at 6.7. Theculture is mixed with an equal volume of 20% MSK solution. The pH of theresulting culture is then lowered to 5.2 by the addition of lactic acidbefore the culture is spray-dried to give the additive containing activedextran sucrase in powder form.

1 l of a milk product containing 2.8% of fats and supplemented with 2%of skimmed milk powder and 6% of sucrose is also prepared, it ispasteurized at 96° C. for 30 min and its temperature is then lowered to42° C.

In a parallel operation, a frozen preculture of a non-thickening strainof Streptococcus thermophilus and a frozen preculture of a non-viscousstrain of Lactobacillus bulgaricus are reactivated in a sterile MSKculture medium containing 10% of reconstituted milk powder and 0.1% ofcommercial yeast extract.

The pasteurized milk product is inoculated with 1% of each of thecultures of the two strains and the milk product is then incubated at40° C. until the pH reaches a value of 4.5. 1% of the additivecontaining the active dextran sucrase is then added in powder form, withstirring.

Yoghurts are produced in this way and stored at 4° C.

These yoghurts, prepared with the additive containing active dextransucrase in powder form, have an unctuous texture with a pleasant taste,especially after storage for 10 days at 4° C.

Example 4

The additive containing active dextran sucrase according to the presentinvention is used in powder form for the manufacture of ice creams.

To do this, a culture medium containing 0.5% of yeast extract and atleast 2% of sucrose is inoculated with 1% of a preculture of the strainof Leuconostoc mesenteroides ssp. cremoris. It is allowed to ferment at23° C. for 20 h with the pH being maintained at 6.7. The pH of theresulting culture is lowered to 5.2 by the addition of lactic acid. Theculture is mixed with an equal volume of 20% MSK solution before beingspray-dried to give the additive containing active dextran sucrase inpowder form.

In another operation, 100 l of an ice cream mixture containing 8% offats, 10% of non-fat solids, 14% of sucrose, 3% of glucose syrup DE36-40, 0.3% of emulsifier, monoglyceride and diglyceride are prepared.This mixture has a total dry extract of 35.28%. The mixture prepared inthis way is stirred at 60-65° C. for 20 min, homogenized at 210 bar andat 72° C. (rising homogenization—2 stages), pasteurized at 86° C. for 22sec and then cooled to +4° C. The output of the homo-pasteurization lineis 200 l/h.

The mixture is acidified to pH 5.5 with lactic acid. 1% of the additivecontaining the active dextran sucrase is then added in powder form, withstirring.

The mixture is then matured at +4° C. When mature, the ice cream mixtureprepared with the additive containing active dextran sucrase in powderform has an unctuous texture. It is frozen on a freezer at −5° C., witha 95% volume increase, at a counter pressure of 3 bar and with an outputof 80 l/h. The ice cream is then stored at −35° C. in a hardeningchamber and subsequently at −30° C. or −20° C.

The ice cream prepared with the additive containing active dextransucrase in powder form has a good textural quality and a pleasant taste.The ice cream obtained in this way gives a good sensation in the mouth.The ice cream is smooth and unctuous. After accelerated ageing the icecream preserves a good level of textural quality and of creamy sensationin the mouth. The smooth character of the ice cream is preserved well;this can be explained by the cryoprotective property of the dextrans,which then limits excessive growth of the ice crystals.

Example 5

The additive containing active dextran sucrase according to the presentinvention is used in powder form for the manufacture of ice creams.

To do this, a culture medium containing 0.5% of yeast extract and atleast 2% of sucrose is inoculated with 1% of a preculture of the strainof Leuconostoc mesenteroides ssp. cremoris. It is allowed to ferment at23° C. for 20 h with the pH being maintained at 6.7. The pH of theresulting culture is lowered to 5.2 by the addition of lactic acid. Theculture is mixed with an equal volume of 20% MSK solution before beingspray-dried to give the additive containing active dextran sucrase inpowder form.

In another operation, 100 l of a premix containing 18% of sucrose areprepared. 1% of the additive containing the active dextran sucrase isthen added in powder form, with stirring. The premix is then incubatedat +4° C.

After incubation, the viscosity of the premix has increased. The premixis made up to give a final ice cream mixture containing 8% of fats, 10%of non-fat solids, 14% of sucrose, 3% of glucose syrup DE 36-40, 0.3% ofemulsifier, monoglyceride and diglyceride. This mixture has a total dryextract of 35.28%. The mixture prepared in this way is stirred at 60-65°C. for 20 min, homogenized at 210 bar and at 72° C. (risinghomogenization—2 stages), pasteurized at 86° C. for 22 sec and thencooled to +4° C. At this point the enzyme is totally inactivated. Theoutput of the homo-pasteurization line is 200 l/h.

The mixture is matured at +4° C. and then frozen on a freezer at −5° C.,with a 95% volume increase, at a counterpressure of 3 bar and with anoutput of 80 l/h. The ice cream is then stored at −35° C. in a hardeningchamber and subsequently at −30° C. or −20° C.

The ice cream prepared from a premix incubated with the additivecontaining active dextran sucrase in powder form has a good texturalquality and a pleasant taste. The ice cream obtained in this way gives agood sensation in the mouth. The ice cream is smooth and unctuous. Afteraccelerated ageing the ice cream preserves a good level of texturalquality and of creamy sensation in the mouth. The smooth character ofthe ice cream is preserved well; this can be explained by thecryoprotective property of the dextrans, which then limits excessivegrowth of the ice crystals.

What is claimed is:
 1. A food or cosmetic composition containing activedextran sucrase enzymes which are produced by a strain of Leuconostocmesenteroides ssp. cremoris and which synthesize sucrose into a dextrancomposition.
 2. A composition according to claim 1 wherein the strainproduces, during culturing in DEX medium, a fermented medium which has,upon passage of 100 ml of the fermented medium through a gravityviscometer having a diameter of 25 mm for determination of viscosity inseconds, a viscosity of at least 12 seconds.
 3. A composition accordingto claim 1 wherein the strain does not ferment lactose.
 4. A compositionaccording to claim 2 wherein the strain is selected from the groupconsisting of isolated bacteria strain Leuconostoc mesenteroides ssp.cremoris CNCM I-1692 and isolated bacteria strain Leuconostocmesenteroides ssp. cremoris CNCM I-1693.
 5. A composition according toclaim 1 wherein the composition is a food and the food is a yoghurt. 6.A food or cosmetic composition which contains active dextran sucraseenzymes and which is prepared by a process which comprises steps of:inoculating a medium for culturing bacteria and which comprises sucrosewith a culture of a strain of Leuconostoc mesenteroides ssp. cremoriswhich produces dextran sucrase enzymes to obtain an inoculated culturemedium and incubating the inoculated culture medium at a temperature offrom 25° C. to 35° C. for from 10 hours to 20 hours to obtain afermented medium comprising a substrate and supernatant; isolating thesupernatant from the substrate and recovering the supernatant to obtaina product comprising active dextran sucrase; and incorporating theactive dextran sucrase product obtained into a substance selected fromthe group consisting of a food substance and a cosmetic substance toobtain a composition which contains active dextran sucrase.
 7. Acomposition according to claim 6 wherein, prior to incorporating theactive dextran sucrase product into the substance, the process furthercomprises a step of drying the supernatant to obtain a dried activedextran sucrase product for the incorporating step.
 8. A compositionaccording to claim 6 wherein the culture medium is incubated at a pH offrom 6 to 7.3 and wherein the process further comprises steps of:adjusting the pH of the supernatant to a pH of from 4.9 to 5.7 to obtaina pH-adjusted product; storing the pH-adjusted product at a temperatureof from 0° C. to 10° C. for from 15 hours to 30 hours to obtain a cooledpH-adjusted product; treating the cooled pH-adjusted product to obtain aprecipitate and a supernatant; isolating the precipitate from thesupernatant and obtaining the precipitate to obtain the active dextransucrase product for the incorporating step.
 9. A composition accordingto claim 8 wherein the cooled pH-adjusted product has a temperature offrom 2° C. to 6° C. and is treated by adding an agent selected from thegroup consisting of polyethylene glycol and ammonium sulfate to thecooled pH-adjusted product.
 10. A composition according to claim 8wherein the process further comprises, after isolating and obtaining theprecipitate product and prior to the incorporating step, a step ofdialing the precipitate product to obtain the active dextran sucraseproduct for the incorporating step.
 11. A composition according to claim9 and wherein the process further comprises, after isolating andobtaining the precipitate product and prior to the incorporating step, astep of dialyzing the precipitate product for removing the agent fromthe precipitate to obtain the active dextran sucrase product for theincorporating step.
 12. A composition according to claim 6 or 8 or 10wherein the strain produces, during culturing in DEX medium, a fermentedmedium which has, upon passage of 100 ml of the fermented medium througha gravity viscometer having a diameter of 25 mm for determination ofviscosity in seconds, a viscosity of at least 12 seconds.
 13. Acomposition according to claim 6 or 8 or 10 wherein the strain does notferment lactose.
 14. A composition according to claim 6 or 8 or 10wherein the strain is selected from the group consisting of isolatedbacteria strain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692 andisolated bacteria strain Leuconostoc mesenteroides ssp. cremoris CNCMI-1693.
 15. A composition according to claim 6 wherein the substance ismilk or a food substance which comprises milk.
 16. A compositionaccording to claim 6 wherein the culture medium comprises the sucrose inan amount of at least 2% by weight.
 17. A process for obtaining activedextran sucrase enzymes suitable for use in a food or cosmeticcomposition which comprises the steps of: inoculating a medium forculturing bacteria and which comprises sucrose with a culture of astrain of Leuconostoc mesenteroides ssp. cremoris which produces dextransucrase enuymes to obtain an inoculated culture medium and incubatingthe inoculated culture medium at a temperature of from 25° C. to 35° C.for from 10 hours to 20 hours to obtain a fermented medium comprising asubstrate and supernatant; and isolating the supernatant from thesubstrate and recovering the supernatant to obtain a product comprisingactive dextran sucrase.
 18. A process according to claim 17 wherein theculture medium is incubated at a pH of from 6 to 7.3 and wherein theprocess further comprises steps of: adjusting the pH of the supernatantto a pH of from 4.9 to 5.7 to obtain a pH-adjusted product; storing thepH-adjusted product at a temperature of from 0° C. to 10° C. for from 15hours to 30 hours to obtain a cooled pH-adjusted product; treating thecooled pH-adjusted product to obtain a precipitate and a supernatant;and isolating the precipitate from the supernatant and obtaining theprecipitate to obtain the active dextran sucrase product.
 19. A processaccording to claim 17 wherein the process further comprises, afterisolating and obtaining the precipitated product and prior to theincorporating step, a step of dialyzing the precipitate product.
 20. Aprocess according to claim 17 wherein the strain produces, duringculturing in DEX medium, a fermented medium which has, upon passage of100 ml of the fermented medium through a gravity viscometer having adiameter of 25 mm for determination of viscosity in seconds, a viscosityof at least 12 seconds.
 21. A process according to claim 17 wherein thestrain does not ferment lactose.
 22. A process according to claim 17wherein the strain is selected from the group consisting of isolatedbacteria strain Leuconostoc mesenteroides ssp. cremoris CNCM I-1692 andisolated bacteria strain Leuconostoc mesenteroides ssp. cremoris CNCMI-1693.
 23. A process according to claim 17 wherein the culture mediumcomprises the sucrose in an amount of at least 2% by weight.